LOCUS       sequence-342950-        6308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses E. coli type I-E Cas7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6308)
  AUTHORS   Wimmer F, Englert F, Beisel CL
  TITLE     A TXTL-Based Assay to Rapidly Identify PAMs for CRISPR-Cas Systems 
            with Multi-Protein Effector Complexes.
  JOURNAL   Methods Mol Biol. 2022;2433:391-411. doi: 
            10.1007/978-1-0716-1998-8_24.
  PUBMED    34985758
REFERENCE   2  (bases 1 to 6308)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods Mol
            Biol."; date: "2022"; volume: "2433:391-411. doi"; pages: " 
            10.1007/978-1-0716-1998-8_24"
COMMENT     Sequence Label: pEcCas7
FEATURES             Location/Qualifiers
     source          1..6308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     969..988
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1008..1085)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1291..1310
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1394..1413
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1394..1412
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1413..1437
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             2566..2583
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2636..2654)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2650..2697
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2734..3189
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2821..2840)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3031..3052
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3281..4096)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3281..4096)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4004..4023
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4088..4107
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4218..4806
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4707..4726
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4960..4977
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4992..5131
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5057..5076)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5217..5239
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5233..5424)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6233..6308,1..1007))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
       61 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
      121 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
      181 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
      241 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
      301 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
      361 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
      421 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
      481 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      541 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      601 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      661 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      721 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      781 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      841 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      901 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      961 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     1021 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     1081 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     1141 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     1201 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     1261 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     1321 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     1381 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1441 ctagactcaa gaaatcacgc aaaggaggta tttataatgt ccaattttat caacattcat
     1501 gttctcatat cccactcgcc ctcgtgcctt aaccgggacg atatgaatat gcagaaagat
     1561 gccatctttg ggggcaaacg cagagttcgt attagctcac agtccctgaa acgtgcaatg
     1621 cgtaaatcag gctattatgc gcagaatatt ggagagagca gcttacgcac cattcacctg
     1681 gcacaattac gtgatgtact gcgtcaaaaa ctgggtgaac gttttgacca gaaaattatt
     1741 gataaaactc tggcactact tagcggtaag tctgtggatg aagcagaaaa gattagcgcc
     1801 gatgcggtga ccccgtgggt ggtaggtgaa atcgcgtggt tttgtgagca ggttgcgaaa
     1861 gcagaagcgg ataatcttga tgacaaaaaa ttgctgaaag tgctgaaaga agatatagcg
     1921 gcaattcgcg ttaatctgca gcagggcgta gatattgccc tgagtggtcg catggccaca
     1981 tcaggtatga tgaccgagtt gggtaaagtc gatggggcga tgtcgattgc acacgctatt
     2041 acaacgcatc aggttgactc cgatatcgat tggtttactg cggtggatga cctgcaggaa
     2101 caagggagcg ctcacttggg tacacaggaa ttttcttcag gcgtttttta tcgttatgca
     2161 aatatcaatc tggctcagtt acaagaaaat ctgggcggcg cgagtagaga acaagctctg
     2221 gaaattgcaa ctcatgtagt tcacatgttg gccacggaag ttccaggggc aaaacagaga
     2281 acctatgcag ccttcaatcc agctgacatg gttatggtga actttagcga tatgcccttg
     2341 agtatggcaa atgcatttga aaaggcagta aaagcgaaag acggttttct gcaaccatca
     2401 attcaagcat ttaatcagta ttgggatcgt gtagcaaacg gatatggtct gaatggtgca
     2461 gctgctcaat tcagtctgag cgatgtggat ccgattacgg cccaagtcaa gcagatgcca
     2521 acgttggaac aactaaaaag ttgggttcgt aataacgggg aagcacacca ccaccaccac
     2581 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     2641 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     2701 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     2761 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2821 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2881 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2941 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     3001 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     3061 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     3121 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     3181 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3241 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     3301 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     3361 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     3421 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     3481 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     3541 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     3601 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     3661 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     3721 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     3781 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     3841 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     3901 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     3961 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     4021 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     4081 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     4141 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4201 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4261 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4321 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4381 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4441 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4501 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4561 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4621 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4681 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4741 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4801 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4861 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4921 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4981 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     5041 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     5101 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     5161 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5221 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     5281 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     5341 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     5401 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     5461 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     5521 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     5581 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     5641 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     5701 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     5761 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     5821 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     5881 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     5941 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     6001 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     6061 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     6121 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     6181 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     6241 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     6301 gagaggcg
//
